The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we are using a unique approach in which the centrosome is first labeled in living cells (by expressing proteins tagged with Green Fluorescent Protein), so that all or just specific parts of it can then be destroyed by laser microsurgery during different stages of the cell cycle. With this method we can create cells that possess this organelle. During the previous grant period we made two important findings relevant to the current proposal. First we found that, contrary to longstanding dogma, the centrosome is not required for formation of the mitotic spindle in vertebrates. Next we demonstrated that the presence of the centrosome is required for cells to progress through the cell cycle during interphase. The research proposed here represents a natural progression from our previous findings, and address the following questions: Aim#1 is to ablate just one centriole to determine if progression through the cell cycle requires the whole centrosome. In Aim#2 we will determine the exact point during G1 at which the presence of the centrosome is required for cell-cycle progression (e.g., before or after midbody separation). Aim#3 is to determine if a centrosome can form de novo after destroying the existing centrosome with the laser and, if so, whether the cell resumes the cell cycle. Aim#4 is to test the hypothesis that epigenetic inheritance of extra centrosomes by a normal diploid cell leads to formation of aneuploid, but viable, progeny and ultimately to a population of transformed (cancerous) cells. Finally, Aim#5 is to determine if mitotic spindle can be formed via centrosome-independent pathway even in the presence of centrosomes in somatic cells. Together the knowledge obtained from these studies will define how the centrosome is involved in cell proliferation and cell cycle control. Since centrosome abnormalities are a hallmark of cell transformation and cancer, this knowledge will be useful for designing new strategies for the treatment of this disease.